Lyme borreliosis, Lyme disease, Lyme arthritis, Bannwarth's syndrome, or erythema chronicum migrans (ECM) are synonymous designations for a zoonotic spirochetal infection transmitted by the bite of ticks of the genus Ixodes. Although the disease was known in Europe for quite some time, it was not discovered in the United States until a 1975 arthritis epidemic occurred in Connecticut. A presumptive etiological agent was isolated from a tick in 1982 (Burgdorfer et. al, Science 216:1317-1319, 1982). In 1984, the spirochete was shown to be a member of the genus Borrelia, and formally named Borrelia burgdorferi (Johnson et. al, Int. J. Syst. Bacteriol. 34:496-497, 1984). As such, it is an evolutionary relative of Borrelia hermsii, Borrelia turicatae, Borrelia anserina, and other members of this arthropod associated genus of spiral bacteria.
Lyme disease is a serious chronic borrelial infection characterized by a diversity of symptoms at various stages. Approximately 3 to 14 days following the initiating tick bite, symptoms may include fever, flu-like illness, and the appearance of the ECM skin rash. Stage two, occurring weeks to months after the initial bite includes further skin involvement, arthritis, nervous system complaints, and cardiac pathology. Stage three is characterized by more severe arthritis and nervous system complications.
Diagnosis of Lyme disease is generally either differential or dependent on host antibody response. Isolation and culture of these bacteria as a diagnostic method is not considered technically or economically feasible. Differential diagnosis relies on time of year (tick season), residence in an endemic area, recollection of tick-bite history, and ECM rash. This diagnostic scheme suffers, respectively, from the chronic nature of the disease, infection of travellers, the incredibly small size of the ticks, and that not all patients experience the ECM rash. Diagnosis based on antibody response requires the seroconversion of infected individuals toward production of anti-B. burgdorferi antibodies. While the antibody approach works for many diagnostic problems, it fails for Lyme diagnosis for the following reasons:
The combination of low numbers of spirochetes and antigenically bland outermembrane yields weak host immune response. PA1 Seroconversion takes some period of time after infection. During the height of spirochetemia, within weeks of the initial tick bite, a patient presenting with fever and ECM may fail to evince antibody. PA1 The antibody response is transitory. During Stage two or Stage three, when circulating bacteria are fewest, antibody titers may be very low. PA1 Cell mediated immunity may detract from circulating antibody signal, that is, classical host IgG and IgM response is decreased or absent due to masked antigens. PA1 Most of the antibody tests rely on whole Borrelia burgdorferi preparations as target antigens, The major problem with this approach is cross-reactivity with an immunological response to other bacterial challenges. Most noteworthy is cross-reaction with anti-Treponema pallidum (syphilis) antibodies. PA1 a) increased sensitivity; i.e., the ability to detect said bacteria in a given sample more frequently; PA1 b) potentially significant reductions in assay cost due to the use of inexpensive reagents and reduced labor; PA1 c) accurate identification of even biochemically unusual strains of the target bacteria, or bacteria with dramatically different outer membrane proteins; PA1 d) direct assay for the presence of the bacterium and consequent potential to quantify the etiological agents; PA1 e) assay independent of the host's immune response schedule--much earlier detection is possible; PA1 f) direct testing allows the monitoring of the efficacy of an antibiotic regime; PA1 g) potential to detect said etiological agent in samples of tissue normally low in antibody titer such as skin.
It is an aspect of the present invention to provide non-immunologically based assays thereby avoiding associated problems and to provide nucleic acid probes which are specific for tick-borne spirochetes capable of causing Lyme disease and related morbidity.
It is another aspect of the present invention to provide probes and assays particularly specific for the type strain of the species, Borrelia burgdorferi B31, American Type Culture Collection strain number 35210.
It is yet another aspect of the present invention to provide nucleic acid probes which can hybridize to target regions which can be rendered accessible to probes under normal assay conditions.
While Kohne et al. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences, they do not provide the teaching necessary to make Borrelia burgdorferi specific probes or, in fact, any other probes to detect spirochete bacteria.
Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels, Similarly, Sogin, Sogin and Woese (Journal of Molecular Evolution 1:173-184, 1972) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships. Fox, Pechman and Woese (International Journal of Systematic Bacteriology 27:44-57, 1977) discuss the comparative cataloging of 16S ribosomal
RNAs as an approach to prokaryotic systematics. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to Lyme spirochetes and in particular, do not provide Lyme spirochete specific probes useful in assays for detecting Lyme disease or its etiological agent, Borrelia burgdorferi and relatives. Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Ribosomes contain three distinct RNA molecules which, at least in Escherichia coli, are referred to as 5S, 16S and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and this convention will be continued herein. An additional convention used herein designates sequence position numbers analogous to those of the Escherichia coli 16S rRNA sequence (Brosius et al., PNAS (USA) 75:4801-4805, 1978).
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that alloy them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially, see next paragraph) to target nucleic acid sequences. In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g. by end capping), to carry detection ligands (e.g. fluorescien, 32-P, biotin, etc.), or to facilitate their capture onto a solid support (e.g., poly-deoxyadenosine "tails"--see FIG. 1). Such modifications are elaborations on the basic probe function which is its ability to usefully discriminate between target and non-target organisms in a hybridization assay.
Hybridization traditionally is understood as the process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion (one oriented 5' to 3', the other 3' to 5') to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another. The high specificity of probes relies on the low statistical probability of unique sequences occurring at random as dictated by the multiplicative product of their individual probabilities. These concepts are yell understood by those skilled in the art.
The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids.
Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar.